WO2016089178A1 - Dispositif et procédé d'induction de cellules pluripotentes utilisant de l'énergie - Google Patents

Dispositif et procédé d'induction de cellules pluripotentes utilisant de l'énergie Download PDF

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WO2016089178A1
WO2016089178A1 PCT/KR2015/013269 KR2015013269W WO2016089178A1 WO 2016089178 A1 WO2016089178 A1 WO 2016089178A1 KR 2015013269 W KR2015013269 W KR 2015013269W WO 2016089178 A1 WO2016089178 A1 WO 2016089178A1
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cells
culture
pluripotent
culture medium
physics
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PCT/KR2015/013269
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English (en)
Korean (ko)
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김순학
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가톨릭관동대학교산학협력단
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Priority to CN201580075450.XA priority Critical patent/CN107438668B/zh
Priority to US15/532,032 priority patent/US11773387B2/en
Priority to JP2017549154A priority patent/JP6986968B2/ja
Priority to EP15866086.0A priority patent/EP3241896B1/fr
Priority claimed from KR1020150172501A external-priority patent/KR101798726B1/ko
Publication of WO2016089178A1 publication Critical patent/WO2016089178A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
    • H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range

Definitions

  • the present invention relates to a pluripotent cell induction apparatus and method using energy that can induce pluripotent cells with pluripotent properties through the provision of energy such as ultrasound, laser or heat treatment.
  • Pluripotency is the ability to differentiate into three types of germ layers: ectoderm, mesoderm and endoderm.
  • Pluripotent stem cells are clinically important in disease models and transplantation because they generate any cell or tissue type in the body.
  • iPSCs induced pluripotent stem cells
  • somatic cells somatic cells
  • patient-derived cells the introduction of foreign genetic material or chemicals or small molecules for clinical applications. It should be simple, fast, effective and safe. Recent studies have demonstrated that the interaction between environment and genotype is closely related to gene expression and phenotypic variation in living organisms.
  • the present inventors express a pluripotent characteristic of expressing and undifferentiating the three-germ marker gene consisting of ectodermal, mesoderm and endoderm by providing energy using cellular environment signals in the absence of genes and chemicals.
  • new multi-functional by cells developed a new method for inducing a so-called Physics (p luripotent sp h ere y ielded by ultra s on ic s timulus) cells with the present invention has been completed.
  • the present invention comprises mixing the culture medium and the differentiated cells, providing energy to the mixture to form a spheroid (spheroid) through a certain period of time,
  • the spheroid has a pluripotency property, and provides a method for reverse differentiation from differentiated cells to pluripotent cells.
  • the present invention also provides a culture chamber capable of containing cells and culture medium; And a device disposed on one side of the culture chamber and capable of providing energy to the cells and the culture medium, mixing the differentiated cells and the culture medium,
  • the spheroid provides a pluripotent cell induction device having a pluripotency property.
  • the present invention differentiates in comparison with known induced pluripotent stem cells, while having pluripotent characteristics in differentiated cells through the provision of energy by appropriate ultrasound, laser, or heat treatment, etc., without the introduction of derivatization inducers and chemicals into differentiated cells. It has the effect of inducing new types of pluripotent cells with strong properties.
  • FIG. 1 is a schematic diagram of human Physics cells having pluripotent properties according to the present invention.
  • Figure 2a shows the effect of the ultrasound intensity on human skin fibroblasts
  • a) is a comparison of HDF morphological changes by ultrasound intensity
  • b) is the number of multicellular spheroids generated by ultrasound intensity.
  • 2B shows the effect of ultrasound intensity on human skin fibroblasts, c) live / dead cell assay of sonicated HDF, d) the percentage of live and damaged cells in c).
  • Figure 3a shows the effect of the ultrasonic exposure time under a fixed intensity of 1 W / cm 2
  • a) is a comparison of HDF morphological changes by ultrasonic exposure time
  • b) is the number of multicellular spheroids generated by the ultrasonic exposure time .
  • Figure 3b shows the effect of ultrasound exposure time under a fixed intensity of 1 W / cm 2 , c) live / dead cell assay of sonicated HDF, d) the percentage of live and damaged cells in c) to be.
  • Figure 4 shows the effect of ultrasound intensity on human ESC culture medium, the top is a comparison of the morphological changes of the ultrasound-treated HDF grown in the sonicated medium, the bottom is the number of multicellular spheroids generated above.
  • Figure 5 shows the effect of the ultrasonic exposure time of human ESC culture medium under a fixed intensity of 5 W / cm 2
  • a) is a comparison of the morphological changes of ultrasonic-treated HDF grown in sonicated medium
  • FIG. 6 shows the effects of sonication conditions and culture conditions for producing multicellular spheroids, a) is suspension culture, b) is monolayer culture.
  • Figure 7a shows the size distribution of multicellular spheroids produced by ultrasonic stimulation stars (UC, UM, UCUM) under floating culture conditions, a) is the total size, b) is the distribution of spheroids of 50-100 ⁇ m size .
  • Figure 7b shows the size distribution of multicellular spheroids produced by ultrasonic stimulation stars (UC, UM, UCUM) under floating culture conditions, c) is 100-200 ⁇ m, d) distribution of spheroids of> 200 ⁇ m size to be.
  • Figure 8a shows the size distribution of multicellular spheroids produced by ultrasonic stimulation stars (UC, UM, UCUM) under monolayer culture conditions, a) is the total size, b) is the distribution of spheroids of 50-100 ⁇ m size .
  • Figure 8b shows the size distribution of multicellular spheroids produced by ultrasonic stimulation stars (UC, UM, UCUM) under monolayer culture conditions, c) is 100-200 ⁇ m, d) distribution of spheroids of> 200 ⁇ m size to be.
  • 9A shows the results of RT-PCR analysis comparing expression levels of pluripotent marker genes, OCT3 / 4 (a) and SOX2 (b), of human Physics cells between suspension culture and monolayer culture conditions.
  • 9B shows the results of RT-PCR analysis comparing expression levels of pluripotent marker genes, NANOG (c) and TDGF1 (d), of human Physics cells between suspension culture and monolayer culture conditions.
  • 10A is a confocal laser microscope image of OCT3 / 4 expression levels in suspension culture.
  • 10B is a confocal laser microscope image of OCT3 / 4 expression levels in monolayer culture.
  • 11 shows RT-PCR analysis of pluripotent marker gene expression for 6 days of culture.
  • 16A shows the results of FACS analysis of human ES (H9) cell surface markers.
  • 16B shows FACS analysis of human HDF cell surface markers.
  • 16C shows FACS analysis of human Physics cell surface markers.
  • Figure 17 shows the results of analysis of protein expression by immunocytochemistry (b) with gene expression RT-PCR (a) of pluripotency markers when co-culture of feeder cells and physics cells.
  • 19 is a result of verification of proliferative capacity of human physics cells, a) shows the expression of Ki67, a proliferation marker, b) stained the increased cells using H33342 and PI, and c) according to the culture time. It shows the size of the spheroid.
  • 21A is an immunocytochemical analysis of the expression of trioderm markers from human ES (H9) cells.
  • 21B is an immunocytochemical analysis of the expression of trioderm markers from human Physics cells.
  • FIG. 22 shows immunocytochemical analysis of expression patterns of OCT3 / 4 and triploid markers for 15 days of culture of human Physics cells.
  • FIG. 23 shows SEM images of human Physics cells according to ultrasonic stimulation conditions.
  • FIG. 24 shows the results of live / dead cell analysis of human Physics cells as seen in fluorescence images after sonication and 2 hours of incubation for each ultrasonic stimulation condition.
  • FIG. 25 shows live / kill cell analysis of human Physics cells after formation of multicellular spheroids.
  • 26 is a change of the Ca 2 + concentration of cells by ultrasonic stimulation.
  • FIG. 28 is a fluorescence image of human Physics cells stained with CM-H2DCFDA to analyze the generation of intracellular H 2 O 2 by sonication in FIG. 27.
  • 29 shows the results of analysis of the release of intracellular ATP by ultrasonic stimulation.
  • FIG. 30 shows expression patterns of P2X and P2Y receptors in human Physics cells.
  • FIG. 31 is a confocal microscopy image demonstrating higher infiltration capacity of human Physics cells using Quantum dot 705.
  • FIG. 31 is a confocal microscopy image demonstrating higher infiltration capacity of human Physics cells using Quantum dot 705.
  • 34 is an immunocytochemical result of the expression of three germline markers of in vitro differentiated human Physics cells.
  • Figure 35 shows the results of RT-PCR analysis of neural line gene expression to confirm in vitro differentiation of human Physics cells.
  • FIG. 41 shows tissue immunofluorescence staining results to confirm in vivo differentiation of human Physics cells in mouse testis.
  • Figure 43 shows the effect of the cell culture medium, a) using the ES medium and HDF culture medium, b) is the result of inducing human Physics cells in ES medium and HDF culture medium and confirming the ES marker.
  • 45 shows the results of deriving human Physics cells from patient skin cells. a) shows the change in cell morphology, and b) shows the result of inducing human Physics cells in other cell lines and identifying ES markers and triplets.
  • FIG. 46 shows the results of heat treatment with different energy sources to induce human Physics cells and to identify ES markers and triploid markers.
  • FIG. 47 shows the results of inducing human Physics cells using a laser as another energy source and identifying ES markers and triploid markers.
  • A is the result of confirming GFP expression in mouse Physics spheroid
  • B Is a tile scan picture that combines a wide range of pictures.
  • FIG. 50 shows the results of analyzing the GFP expression rate in all cells treated with ultrasound using the GFP expression rate graph and the flow cytometry of the spheroid formed by ultrasound.
  • Figure 51 shows the results of analysis of the expression of cell surface undifferentiated marker (SSEA1) in mouse Physics cells using flow cytometry.
  • Fig. 53 shows the results of analysis of pluripotent protein markers in mouse Physics cells by immunocytochemistry.
  • FIG. 55 shows the results of RT-PCR analysis of the expression of trioderm markers from mouse Physics cells.
  • Fig. 56 shows the results of immunocytochemistry analysis on the expression of three germ layer markers from mouse Physics cells.
  • the present invention comprises mixing the culture medium and the differentiated cells, providing energy to the mixture to form a spheroid through a certain time of culture,
  • the spheroid relates to a method for dedifferentiating from differentiated cells to pluripotent cells, which has pluripotency properties.
  • the present invention provides pluripotency characteristics from differentiated cells by providing appropriate energy without introducing dedifferentiation inducers or chemicals into the differentiated cells. It is characterized by being able to induce a new type of pluripotent cells with differentiation properties.
  • the pluripotent cells are distinguished from known induced pluripotent stem cells in that differentiation is well induced according to an external environment, and that the properties of progenitor cells having strong differentiation properties compared to those of stem cells are stronger. .
  • a preparatory step of undergoing a differentiation process is required, which includes a risk factor that can turn into cancer, and a virus for introducing a differentiation inducer.
  • pluripotent cells of the present invention are induced without introducing a differentiation-inducing substance such as a back-differentiation inducer or a chemical for genetic variation, so co-culture with other types of cells
  • a differentiation-inducing substance such as a back-differentiation inducer or a chemical for genetic variation
  • the pluripotent cells of the present invention have the advantage that the induction process is simple and short, so that time can be dramatically reduced until transplantation by treating autologous cells.
  • the present invention is specifically capable of producing spheroids in good yields by providing energy to both culture medium and differentiated cells.
  • the energy can be any one of ultrasound, laser or heat treatment.
  • the pluripotent cells of the present invention are any of the undifferentiated markers of OCT3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP It is characterized by stably expressing three germ layer marker genes consisting of mesoderm and endoderm.
  • the inventors considered the association with exosomes with respect to the formation of pluripotent cells without introduction of dedifferentiation inducers into differentiated cells. That is, ultrasound, laser, or heat treatment induces temperature rise by energy, induction of reactive oxygen species (ROS), vibration of microbubbles generated by ultrasound, induction of flow generation of liquid, that is, induction of microstream generation along the cell membrane.
  • ROS reactive oxygen species
  • This effect causes fine damage to the cell membrane and induces the formation of pores to increase the uptake of foreign substances, which is demonstrated by confirming the intracellular Ca 2 + concentration change analysis and H 2 O 2 production.
  • ATP was used as a response signal to various cellular stresses and analyzed the ATP concentration in pluripotent cells after sonication, and released ATP at a higher level than the untreated control group.
  • the expression of ion-directed P2X receptor and metabolic P2Y receptor by ATP release was also activated in pluripotent cells compared to the control group.
  • exosomes are known to contain genetic information (DNA, mRNA, microRNA, protein) inside the exosomes through the process of exosomes that escaped out of the cell membrane through the damage to the cell membrane reenters other cells around the inside Genetic information material present in can be delivered. Therefore, as the stimulation by the ultrasonic treatment causes the expression of undifferentiated markers that have been under-expressed or remained in the suppressed state of the cell and damages the cell membrane, the undifferentiated markers in which the expression is induced or promoted are induced. Exosomes inside the containing cells are discharged to the outside and delivered to the surrounding cells.
  • DNA DNA, mRNA, microRNA, protein
  • the culture medium is recovered during the process of pluripotent cell induction, and the exosomes in the medium are extracted to determine whether there are pluripotent cell-related undifferentiated markers inside the exosomes. It is seen and confirmed to support the hypothesis of the present inventors. In addition, such ultrasonic, laser, or heat treatments were found to be normal without karyotyping.
  • pluripotent cell refers to a cell that has obtained pluripotency after energy, laser, or heat treatment in a strict sense.
  • the pluripotency means a state of stably expressing undifferentiated markers expressed in embryonic stem cells. In addition, it means a state expressing three kinds of endoderm, ectoderm and mesoderm three germ layers markers.
  • the pluripotent cells may be used interchangeably with "Physics (p luripotent sp h ere y ielded by ultra s on ic s timulus) cell", or "Physics spheroid". The differentiation method of the differentiated cells into pluripotent cells of the present invention will be described in detail with reference to FIG. 1 as follows.
  • the cell culture medium and the differentiated cells are mixed and the mixture is energized.
  • Energy may be provided to the cell culture medium prior to providing energy to the mixture of differentiated cells to increase the efficiency of reverse differentiation into pluripotent cells.
  • the energy may be any one of ultrasonic wave, laser, or heat treatment.
  • Ultrasonic treatment of the culture medium has an output intensity of 1W / cm 2 1 to 20 minutes to the ultrasound of 20W / cm 2, specifically, the output strength of 2W / cm 2 To 10 W / cm 2 of ultrasound for 5 to 15 minutes, more specifically, output intensity 3 W / cm 2 To 7 W / cm 2 may be performed for 7 to 13 minutes.
  • Laser treatment of the culture medium may be performed from 1 to 20 seconds for pulsed laser beams in the 300 to 900 nm wavelength band, more specifically 3 to 10 seconds for pulsed laser beams in the wavelength band, more specifically to the wavelengths.
  • the pulsed laser beam of the band may be irradiated for 4 to 6 seconds.
  • the wavelength band may use a wavelength of 400 nm, 808 nm, and 880 nm.
  • Heat treatment of the culture medium may be carried out for 5 to 20 minutes at a temperature condition of 40 to 50 °C.
  • culture medium embryonic stem cell culture medium, stem cell differentiation induction medium and the like can be used.
  • fibroblasts derived from mammals Cancer cells, including cervical cancer cells (HeLa cells); Or organ tissue cells, including lung epithelial cells (L132 cell) can be used.
  • Cancer cells including cervical cancer cells (HeLa cells);
  • organ tissue cells including lung epithelial cells (L132 cell)
  • L132 cell lung epithelial cells
  • the sonication of the mixture of culture medium and differentiated cells is performed for 1 to 5 seconds at a power intensity of 0.5 W / cm 2 to 3 W / cm 2 , more specifically, from 0.7 W / cm 2 to 2 W / cm 2.
  • the output strength may be performed for 1 to 5 seconds at 0.8W / cm 2 to 1.5W / cm 2 .
  • Laser treatment of the mixture of the culture medium and the differentiated cells can be performed from 1 second to 20 seconds for a pulsed laser beam in the 300 to 900 nm wavelength band, more specifically 3 seconds to 10 seconds for a pulsed laser beam in the wavelength band, More specifically, the pulsed laser beam of the wavelength band may be irradiated for 4 seconds to 6 seconds.
  • the wavelength band may use a wavelength of 400 nm, 808 nm, and 880 nm.
  • the heat treatment of the mixture of the culture medium and the differentiated cells may be performed by exposing for 1 minute to 10 minutes at a temperature condition of 40 to 50 ° C. and then exposing for 5 to 10 seconds at a temperature condition of 0 ° C. to 4 ° C. .
  • the mixture provided with energy is incubated for a period of time to form a spheroid having pluripotency.
  • Cultivation of the energy-supplied mixture may be performed for a period of 3 to 10 days during which spheroids stably expressing undifferentiated markers are formed through a suspended culture or monolayer culture.
  • the incubation time is different depending on whether the spheroid formation having pluripotency depending on the culture method, cell or culture medium can be appropriately adjusted at the level of those skilled in the art.
  • the suspension culture exhibits higher spheroid formation efficiency compared to monolayer culture.
  • the floating culture has a larger number and size of spheroids than a single layer culture, and shows a constant size distribution.
  • the expression of undifferentiated markers is increased or stabilized from about 3 days in the suspension culture of human dermal fibroblasts subjected to ultrasound or laser treatment, and dedifferentiation starts from this period.
  • the expression of undifferentiated markers increased or stabilized from about 8 days, and it appears that reverse differentiation starts from this time.
  • spheroids have pluripotency through expression of undifferentiated markers such as OCT3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, and the like. Identification of undifferentiated markers may be analyzed by RT-PCR or immunocytochemistry, but is not particularly limited thereto.
  • the pluripotent cells of the present invention are characterized by expressing high levels of trioderm markers, ie ectoderm (PAX6, Nestin), mesoderm (Brachyury, SMA), endoderm (GATA4, AFP) markers.
  • trioderm markers ie ectoderm (PAX6, Nestin), mesoderm (Brachyury, SMA), endoderm (GATA4, AFP) markers.
  • the pluripotent cells of the present invention are characterized by having proliferative capacity by expressing the proliferation marker protein Ki-67.
  • the co-culture of the dedifferentiated pluripotent cells with feeder cells increases the proliferation of pluripotent cells and differentiates them into ectoderm / endoderm / mesoderm and neurons / cardiomyocytes after culture in differentiation-inducing medium. have.
  • the invention also relates to the invention.
  • a culture chamber capable of containing cells and culture media
  • Differentiated cells are mixed with the culture medium, and energy is supplied to the mixture to form a spheroid through culturing for a period of time, and the spheroid has a pluripotency property.
  • the culture chamber generally means an incubator used for cell culture.
  • the culture chamber is provided with a temperature control unit and a carbon dioxide control unit, the cell culture conditions in the culture chamber can be appropriately adjusted at the level of those skilled in the art according to the purpose and type of cells.
  • the culture chamber may use a floating culture or a monolayer culture method for reverse differentiation from differentiated cells to pluripotent cells, so that the culture chamber may have such a structure.
  • it may be a culture chamber equipped with a stirrer for suspension culture.
  • the device capable of providing energy may include an ultrasonic generator capable of irradiating ultrasonic waves, a laser generator capable of irradiating laser, or a temperature controller.
  • the ultrasonic generator may be used without limitation as long as it is a known ultrasonic apparatus that generates ultrasonic waves having a frequency of 10 kHz to 100 MHz.
  • the laser generator generates a pulsed laser beam having a wavelength range of 300 to 900 nm, and may use a laser device having a pulse duration of 1 ms to 900 ms and a frequency of 1 to 100 Hz with a 1 to 15 W output. It is not limiting.
  • the temperature control device may use a known temperature control device capable of temperature control in the range of -40 ° C to 99.9 ° C, but is not particularly limited thereto.
  • Pluripotent cell induction apparatus of the present invention is a mixture of the culture medium and the differentiated cells using the ultrasonic wave generator, laser generator, or temperature control device, ultrasonic, laser, or heat treatment and pluripotency through a certain time incubation
  • the culture medium may be subjected to ultrasonic, laser, or heat treatment in advance before mixing the culture medium with the differentiated cells.
  • FIG. 1 is a person in Physics (p luripotent sp h ere y ielded by ultra s on ic s timulus) in cells forming a schematic view, intensity of (embryonic stem) ES sonicated for 10 minutes using a 5W / cm 2 culture medium of the present invention skin fiber
  • the blast cells (HDFa, Cat. No. C-013-5C, GIBCO (Invitrogen cell culture) (1 ⁇ 10 6 ) were mixed, and the mixture containing the cells was treated with ultrasound at a intensity of 1 W / cm 2 for 5 seconds. After viable cells were selected, 2 ⁇ 10 5 HDFs were suspended cultured in human ES culture medium for 6 days in a 35 mm bacterial Petri dish.
  • Spheroids are formed from the first day of culture, and undifferentiated markers are expressed from three days later.
  • the cell culture method is suspended culture cultured in an uncoated dish (bacterial petri dish) and cell culture dish surface (cell culture dish) Monolayer culture was used.
  • the group that was not treated as a control group (Null)
  • the group treated with ultrasonic waves in the medium (UM: Ultrasound treated Media, 5W / cm 2 treated with ultrasonic intensity for 10 minutes)
  • the group treated with ultrasonic waves (UC : Ultrasound treated Cell, treated with ultrasonic intensity of 1W / cm 2 for 5 seconds)
  • UCUM Ultrasound treated Cell, treated with ultrasonic intensity of 1W / cm 2 for 5 seconds
  • UCUM Ultrasound treated Cell, treated with ultrasonic intensity of 1W / cm 2 for 5 seconds
  • the ultrasonic intensity (0, 0.5, 1, 3, 5, 10 W / cm 2 for 5 seconds) was directly exposed to HDF (1 ⁇ 10 6 ). After viable cells were selected, 2 ⁇ 10 5 HDFs were cultured in human ES culture medium for 6 days in a 35 mm bacterial Petri dish.
  • ES medium composition Reagent Name Volume Final concentration Remarks DMEM / F-12 500 mL 500 mL Serum Replacement (KnockOut TM Serum Replacement) 100 mL 20% NEAA (non-Essential Amino Acids) 5 mL One% P / S (Peniciline & Streptomycin) 5 mL One% ⁇ -Mercaptoethanol 0.9 mL 0.1mM Glutamin (L-Glutamine, 200 mM Solution) 2.5mL 1 mM basic human fibroblast growth factor 2 (FGF) (Recombinant Human FGF-Basic) 2mg 4ng / mL Addition after sonication
  • FIG. 2A (b) shows the number of multicellular spheroids generated for each ultrasound intensity in FIG. 2A (a).
  • the ES cell culture medium was treated for 10 minutes with different ultrasonic exposure intensities (0, 1, 5, 10 W / cm 2 ).
  • Ultrasonically exposed 2 ⁇ 10 5 HDF (1 W / cm 2 , 5 sec) in a 35 mm bacterial Petri dish was incubated in these media for 3 days.
  • ESC culture medium was treated with ultrasound (5 W / cm 2 , 10 minutes)
  • HDF (1 ⁇ 10 6 ) was treated with ultrasound ( 1 W / cm 2 , 5 seconds).
  • Live HDF ( ⁇ 10 5 ) was screened and suspended in bacterial petri dishes or monolayer culture in tissue culture dishes.
  • suspended cultured ultrasound-treated HDFs showed higher spheroid formation efficiency compared to monolayer cultures.
  • Ultrasonic stimulation showed higher spheroid formation efficiency when both cells and culture medium were stimulated.
  • sonicated HDF or untreated HDF was sonicated in a bacterial petri dish or tissue culture dish in an ES cultured medium sonicated or untreated. Incubated.
  • the floating culture conditions showed higher efficiency, and the spheroids were larger in number and size (diameter of 200 ⁇ m or more) and showed a constant size distribution than the monolayer culture conditions.
  • Sonicated HDF (UC) grown in untreated ES cell culture media formed spheroids.
  • the number and size of spheroids up to 200 ⁇ m
  • Normal HDF culture (UM) in sonicated ES cell medium resulted in the formation of small amounts of spheroids (less than 100 ⁇ m).
  • UC and UM conditions of monolayer culture using tissue culture dishes were too low for spheroid formation efficiency.
  • Most HDFs were attached to the culture plate surface and the spheroid count was too small.
  • control and ultrasonic treatment groups (Null, UM, UC, UCUM) cells were harvested for each incubation time (1, 2, 3, 4, 5 and 6 days), mRNA was extracted using Dynabeads® mRNA direct kit (ambion), and SuperScrip-II (invtrogen) cDNA was synthesized. It was then amplified by PCR using the primers described in Table 2 and analyzed by electrophoresis.
  • the expression of the undifferentiated marker gene was stably expressed when the HDF and the culture medium were both sonicated (UCUM) in FIG. 9, and the suspension culture was higher than that of the monolayer cultured cells.
  • the expression level of the OCT3 / 4 which is an undifferentiation marker, was compared in the suspension culture or the monolayer culture.
  • cells cultured for 4 hours (0, 1, 2, 3, 4, 5 and 6 days) after sonication were fixed with 4% paraformaldehyde for 30 minutes, and 0.1% to improve the penetration ability of the antibody.
  • blocking was performed for 30 minutes at room temperature with PBS buffer containing 5% non goat serum to prevent nonspecific protein reactions.
  • each of the primary antibodies OCT4; 1: 200, abcam
  • OCT4 was added and reacted at 4 °C overnight, washed three times with PBS buffer added 0.03% Triton X100 secondary antibody (IgG
  • the anti-rabbit conjugate alexa 488) was diluted 1: 1000 with D-PBS buffer and stained at room temperature for 2 hours. Stained cells were washed four times using PBS buffer with 0.03% TritonX100, then covered with slides with DAPI-added mounting sol. Observed by.
  • OCT3 / 4 expression was detected immediately after 1 day of sonication in UCUM conditions. OCT3 / 4 expression gradually increased, and floating culture conditions showed higher expression levels than monolayer culture conditions.
  • OCT3 / 4 six differentiation marker genes, OCT3 / 4, SOX2, NANOG, TDGF1, c-MYC and KLF4, were analyzed by RT-PCR for 6 days of spheroid culture under UCUM in suspension culture.
  • the expression of OCT3 / 4 and NANOG genes increased on day 1 after treatment, and the expression of other genes also increased with incubation time. All marker genes were observed on day 2 but stable expression was observed. Was observed after 3 days.
  • the first OCT3 / 4 expression time point was detected immediately 10 hours after sonication (FIG. 12).
  • the experiment was performed by harvesting physics cells cultured for 5 days, extracting mRNA using Dynabeads® mRNA direct kit (ambion), synthesizing SuperScrip-II (invtrogen) cDNA, and amplifying by PCR using the primers described in Table 2. It was analyzed by electrophoresis.
  • DNA methylation analysis was performed to further confirm the expression of the undifferentiated marker.
  • the promoter portion where the gene expression starts is methylated it can be seen that the gene is not expressed in that portion, which means that the gene of the portion is expressed when demethylated, that is, when the methyl group is separated from the DNA. Therefore, we examined whether the expression of the OCT3 / 4 and NANOG genes, which are the major genes of ES cells, are occurring and whether the promoter portion of the two genes is methylated.
  • DNA amplification primers used in the analysis are as follows.
  • Reverse primer 5'-ATCTATCCCTCCTCCCAAATAATC-3 '
  • Amplification product size 377 bp, Tm: 55, CpGs from product: 6
  • Reverse primer 5 / -AATTACAAAAACCATACCTACAACC-3 '
  • Amplification product size 417bp, Tm: 55, CpGs from product: 4
  • CpG cytosine guanine dinucleotide
  • Ki-67 immunostaining method which is a proliferation marker protein, and time-phase nuclear nuclear staining using pst (Hoechst 33342) and propidium iodide (PI).
  • a live / dead kit was added to the sonicated HDF, and the green / red double-stained HDF was added to the live cell imaging device for 24 hours. was followed.
  • Ultrasound-induced membrane damage and intermittent transmission is also within the intracellular Ca 2 + concentrations and H 2 O 2 respectively increased by using the fluorescent dye Fluo-4 dye and CM-H2DCFDA cells Characterized by production.
  • the Ca + 2 concentration of Physics cells Upon exposure to ultrasonic waves, the Ca + 2 concentration of Physics cells and then abruptly increased, decreased to 150 seconds (FIG. 26).
  • the concentration of intracellular H 2 O 2 in Physics cells was six times higher after 60 minutes of ultrasound exposure compared to untreated control HDF (FIGS. 27 and 28).
  • Ionic oriented P2X receptors and metabolic P2Y receptors are known to be activated by ATP release and compared the expression of these receptors.
  • QD705 Alexa-705 labeled quantum dots
  • exosome RNA was prepared from Physics cell culture medium, and the gene expression pattern of the cell culture environment during the generation of Physics cells by RT-PCR analysis was studied.
  • exosomes include several genetic elements, such as RNA, microRNA, DNA, proteins.
  • the expression profile of genetic elements in exosomes is cell state-dependent.
  • pluripotent marker genes As shown in FIG. 32, high expression of pluripotent marker genes was observed in exosomes purified from Physics cell culture medium. The most prominent gene expressions were OCT3 / 4 and NANOG. As the incubation time progressed, OCT3 / 4 expression increased significantly. NANOG expression dropped after 4 days. c-MYC expression was constant in suspended culture conditions, whereas decreased after 2 days in monolayer culture conditions. Expression of all pluripotent marker genes, such as REX1, TDGF1, FOXD3, UTF1, LIN28, was detected in suspension culture conditions, although their expression levels were low. However, these genes were not detected in monolayer culture conditions. These results suggest the possibility of delivery of genetic elements in sonicated HDF.
  • Type of badge ingredient content Medium 1 (ectoderm / astrocytic differentiation induction medium) DMEMFBSN2 supplementGlutamax-I 1% 1% 1% Medium 2 (mesoderm / cardiomyocyte differentiation induction medium) DMEMFBS2-mercaptoethanolNon essential amino acid Penicillin / streptomycin Ascorbic acid 20% 1% 1% M 100 ⁇ M Medium 3 (endodermal / nerve cell differentiation induction medium) DMEMFBS2-mercaptoethanolNon essential amino acid Penicillin / streptomcin 20% 1% 1%
  • gene 17 (SOX17, endoderm), paired box 6 (PAX6, ectoderm), Nestin (nerve cell marker), microtubule-associated protein 2 (MAP2, Ectoderm), class III beta-tubulin (TuJ1, neuronal marker), msh homeobox 1 (MSX1, mesoderm), Brachyury (mesoderm), myosin light chain 7 (MYL7, cardiomyocytes), NK2 homeobox 5 (NKX2.5, myocardium) Cells), and expression of SRY-box including Troponin T type 2 (TnnT2, cardiomyocytes) was observed by RT-PCR.
  • neuroprogenitor markers (PAX6 and Nestin) were observed in Physics cells grown in astrocytic medium. These differentiated Physics cells expressed expression of oligodendrocyte markers (MAP2 and O4) or neuron markers (MAP2 and Tuj1), respectively, when further differentiation was induced for 2 weeks after changing astrocytic medium to oligodendrocyte or neuronal medium. This was observed. Two weeks of differentiation were sufficient to detect cardiac markers including MHC, SMA, Actinin, NKX2.5 and TnTc. In particular, a typical segmented actin pattern was detected in actinin. However, under the same culture conditions, HDF did not express any nerve or heart markers.
  • MAP2 and O4 oligodendrocyte markers
  • Tuj1 neuron markers
  • Fig. 41 the physics cells injected into the testis were observed in vascular endothelial cells in the testis after 4 weeks, and were confirmed to be proliferated by Ki67 staining. As shown in the cells indicated by the arrows, the vascular endothelial marker CD44 was stained It was confirmed.
  • ES cell culture media were used to generate Physics cells.
  • ES cell culture medium was developed as a restriction medium for maintaining and propagating ES cells in an undifferentiated state.
  • normal HDF culture medium was used to generate Physics cells.
  • morphology and spheroid formation efficiency were quite different compared to ES cell culture media.
  • One day after seeding in sonicated HDF medium small amounts of multicellular spheroids were formed. However, after two days, the majority of spheroids were attached to the surface of the dish. On day 4 of culture all spheroids adhered to the surface of the dish and grew into a typical fibroblast form.
  • Immunohistochemical results also showed different gene expression patterns between the two different culture medium conditions.
  • Typical Physics cells generated using ES cell culture media showed high expression levels of OCT3 / 4, SOX2, NANOG, SSEA-4, and TRA-1-60.
  • DMEM media did not show any effect to induce undifferentiated marker gene and trioderm marker gene expression.
  • two cell lines also formed multicellular spheroids after being cultured in ES cell culture media sonicated in bacterial Petri dishes after direct exposure of ultrasound.
  • the shape and size distribution of the new Physics cells from the two cell lines are quite different.
  • Physics cells derived from HeLa cells were quite inconsistent and too large in size.
  • Physics cells derived from L132 cells were more complex and aggregated. Each spheroid was further fused to form a plate-like structure.
  • pluripotency markers including OCT3 / 4, SOX2, NANOG, SSEA4, and TRA-1-60 and GATA4, AFP, PAX6, Nestin, Brachyury, and from two different Physics cells Expression of trioderm marker genes, including SMA, was confirmed by immunocytochemistry.
  • the skin fibroblasts were exposed to 42 ° C. for 2 minutes and then left on ice for about 5 seconds.
  • both HDF and ES cell culture media successfully developed multicellular spheroids after laser or heat treatment.
  • Laser treated HDF also immediately formed multicellular spheroids after laser induction.
  • the shape of the spheroids was irregular and the size distribution was not uniform, high levels of expression of pluripotency markers and triploid markers were observed.
  • Heat treatment also induced spheroid formation.
  • the efficiency was lower than ultrasonic and laser treatments. More than half of the heat-induced multicellular spheroids adhered to the dish surface for 8 days of maintenance. Despite the lower spheroid formation efficiency, higher expression levels of pluripotency markers and triploid markers were observed.
  • Mouse Physics cells were prepared according to the procedure shown in FIG. 48. To this end, OG2 mouse MEF (Mouse Embryonic Fibroblast cells; mouse embryonic fibroblasts) was mixed with ES medium treated with 20 KHz ultrasound at a intensity of 5 W / cm 2 for 10 minutes, and the ultrasound was directly applied to the cells for 1 second at 5 W / cm 2. Incubated by treatment at the strength of. The cultured cells were observed for morphological changes and expression of GFP fluorescence by fluorescence microscopy at 1, 3, 5, 8 and 10 days intervals. The medium composition for the ultrasonic treatment is shown in Table 1.
  • OG2 mouse MEF Mae Embryonic Fibroblast cells; mouse embryonic fibroblasts
  • the MEF cells are fibroblasts of 13.5-day-old embryos of mice transfected with the GFP gene into which the OCT4 promoter has been inserted. Generally, cells expressing OFP4 do not express GFP.
  • the control group did not show green fluorescence on OG2 MEF cell photographs (no OCT4 expression).
  • OCT4 is a major feature of undifferentiated stem cells, which indicates that OG2 MEF cells are reversely differentiated into stem cells by the ultrasonic treatment.
  • FIG. 49B is a result of showing the ultrasonic treatment effect by combining a wide range of pictures with tile scan photos, and a large number of MEF cells express OCT4-GFP due to the differentiation caused by the ultrasonic treatment, and generated in FIG. 50.
  • the OCT4-GFP expression efficiency was about 93%, and GFP expression was expressed in 85.3% of the cells by using flow cytometry.
  • SSEA1 a cell surface undifferentiated protein marker, expression of about 75.5% was shown (FIG. 51).
  • mPhysics expressed high levels of markers of endoderm (GATA6), ectoderm (Nestin) and mesoderm (Brachyury). Expression of other genes in the germ layers began three days after the production of Physics cells. During 20 days of culture, the expression level of the trioderm markers gradually increased (FIG. 55). In addition, expression of trioderm protein markers was confirmed through immunostaining in FIG. 56.
  • mPhysics cells formed by ultrasound had a normal karyotype (FIG. 57).
  • Pluripotent cells of the invention can be used in the field of cell therapy.

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Abstract

La présente invention concerne un dispositif et un procédé d'induction de cellules pluripotentes utilisant de l'énergie et, plus spécifiquement, présente un effet d'induction de cellules pluripotentes d'un nouveau type ayant des caractéristiques pluripotentes en appliquant de l'énergie telle que des ondes ultrasonores, des lasers ou un traitement thermique à des cellules différenciées.
PCT/KR2015/013269 2014-12-04 2015-12-04 Dispositif et procédé d'induction de cellules pluripotentes utilisant de l'énergie WO2016089178A1 (fr)

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CN111073881A (zh) * 2018-10-02 2020-04-28 金贤锡 用于毛发再生的含有诱导的外泌体的组合物

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US20180127738A1 (en) * 2016-11-07 2018-05-10 BiomediStem, LLC Production and therapeutic uses of epinul pluripotent cells and differentiated cells derived therefrom
EP3555262A4 (fr) * 2016-11-07 2020-05-13 K2 Research Holdings, LLC Production et utilisations thérapeutiques de cellules pluripotentes d'epinul et cellules différenciées dérivées de celles-ci
CN111073881A (zh) * 2018-10-02 2020-04-28 金贤锡 用于毛发再生的含有诱导的外泌体的组合物

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